Fig. 3.

Structural analysis of ISG15 specificity. (A, Left) The Lb^pro∼ISG15^CTD-ΔC complex is shown as in Fig. 2D. (A, Right) Close-up view of the hydrophobic contact between Lb^pro (Leu92, Pro99, Leu102) and ISG15 (Trp123) (Fig. S4). (B) ISG15-TAMRA cleavage assays with Lb^pro point mutants in the hydrophobic patch. (C) Lb^pro cleavage assays with WT and W123A proISG15^CTD. (D, Left) Model of ubiquitin (Protein Data Bank ID code 1ubq) bound to Lb^pro. Ubiquitin (red) was superimposed onto ISG15^CTD. (D, Right) Close-up view of the analogous interface as in A. (E) Met1 diubiquitin cleavage assays with or without R42W mutation in the distal ubiquitin. Arrows in the schematics show the proteolytic site of cleavage (Fig. S4E). Assays in B, C, and E were performed in triplicate.